As optical microscopy allows in vivo imaging, it has become one of the most important tools in biological research and technical advances are continually pushing the limits of microscope performance and versatility. In recent years new approaches have been proved to produce super resolution. Examples of these new strategies include Stimulated Emission Depletion Microscopy (STED), Photo Activation Localization Microscopy (PALM) or Stochastic Optical Reconstruction Microscopy, STORM) and structured illumination.
Super resolution is defined as microscopy which allows imaging at a resolution beyond the diffraction limit of the incident electromagnetic radiation. In STED, the diffraction limit is overcome by producing an effective nanoscale fluorescent spot. This is done by depleting electronic excited states via stimulated emission in the peripheral region of a focused fluorescent spot while leaving a small fluorescent region in the centre intact.
STED was the first concrete and feasible concept which showed that the diffraction limit could be exceeded in fluorescence microscopy. Theoretically proposed in 1996 and experimentally demonstrated in 1999, STED microscopy is based on the reduction in the size of the effective focus by switching off the fluorescence ability of the fluorophores in the outer part of the excitation focus. To accomplish this, two spatial overlaid beams with different wavelength are necessary. The first one, called the excitation beam excites the fluorophore molecules within the light path from the ground state to the first electronically excited stated. The second one, called the depletion beam or STED beam, empties the excited states of the fluorophore through stimulated emission in such a way that the fluorophore is switched off. Importantly, the shape of the focused STED beam may be modified to obtain substantially zero intensity at the centre and high intensities at the periphery. This shape can be created, for example, by applying a vortex phase plate. In this way, after excitation, the depletion beam switches-off all the fluorophores except those at the centre, restricting the effective fluorescence generation to a sub-diffraction-sized volume.
The higher the applied STED intensity, the more efficient the depletion is and thus the smaller the central region where the fluorescence is unaltered. Thus, in diamond colour centres, resolutions as small as 6 nm have been demonstrated. On biological samples, resolutions are typically in the 40 to 90 nm range.
STED relies on several fluorophore parameters such as high cross section for stimulated emission, no excitation at the depletion wavelength and photo-stability at depletion and excitation wavelengths. This means that for the currently available dyes, STED lasers which are able to produce a beam at the appropriate wavelength and having sufficient intensity are required. For example, in the earlier designs, pulse sources such as Ti:sapphire lasers producing high peak powers were used. Under this scheme, the use of STED microscopy was limited to the dyes able to be depleted in the 700 nm region (Pyridine3, RH-41411, Atto647N9, Atto655). In an effort to access other visible fluorescent markers, Ti: sapphire lasers or regenerative amplifiers where combined with frequency-doubled optical parametric oscillators (OPO) (RegA) and optic parametric amplifier (OPA). The use of super continuum laser sources has also allowed STED imaging at different colours.
Other novel light sources based on a comb-like spectrum generated in standard single-mode fibres via stimulated Raman scattering (SRS) have been proposed. These SRS light sources enable multicolour STED from green to red (530 and 616 nm). More recently CW lasers have become available with sufficient power for STED microscopy. CW lasers simplify the STED microscope setup considerably because temporal synchronization of the STED pulses with their excitation counterparts becomes obsolete. Thus, a CW STED system has been recently commercialized using a fibre laser with a wavelength of 592 nm and a power of 1.5 W. Under this configuration super resolution on samples with visible fluorescent dyes (Alexa488, Chromeo488, Atto488, FITC) and fluorescent proteins (eYFP, Citrin, Venus) can be obtained.
Typically STED images can be obtained from planes 10 to 15 μm inside the sample while in confocal microscopy, this can go up to 200 μm. This is because sample aberrations produce distortions of the STED beam compromising the super resolution capability at those the penetration depths.
STED microscopy has been used to demonstrate the concept of super resolution imaging in living samples. For instance, S. cerevisiae yeast cells were one of the first organisms imaged with STED microscopes. Since then more complex organisms and their components have been observed using different STED configurations. These include different cytoskeletal structures (tubulin and vimentin), endoplasmic reticulum, protein clusters on the cell membrane in mammalian cell dendritic structures and synaptic vesicles in movement.
It should be noted that standard STED is an intrinsic 2D super resolution technique. This is achieved only on the transversal plane while the axial resolution is limited to that of a conventional confocal microscope. STED has been shown to produce 3D super resolution images by introducing an extra arm with an additional phase mask that introduces a π phase shift in a central disk. However, these phase masks are wavelength dependant and the setup requires the use of two cross polarized beams that have to be combined in an interferometer-based arrangement. This adds complexity and seriously compromises the stability and versatility of the system.